Aquaculture Nutrition

2015
..........................................................................................
doi: 10.1111/anu.12302

1 1 1 2
1
School of Animal Production Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon
Ratchasima, Thailand; 2 Department of Biology, Faculty of Science, Naresuan University, Muang, Phitsanulok, Thailand

Probiotics are defined as (live) micro-organism feed supple-

The goal of this study was to investigate the effects of
ments that beneficially influence host animals by healthy
dietary supplementation with b-glucan and microencapsu-
modulation of intestinal microbiota, conferring benefits on
lated probiotics (Bacillus subtilis or Pediococcus acidilactic-
growth performance, efficiency of feed utilization, modulat-
i) on growth performance, body composition,
ing digestive enzyme activities, survival rate and water
haemolymph constituents, and intestinal morphology and
quality (Fuller 1989; FAO/WHO 2001; Luis-Villase~ nor
microbiota of the Pacific white shrimp Litopenaeus vanna-
et al. 2013). In aquatic animal culture, probiotics such as
mei. Four treatment diets [basal diet (C), b-glucan-con-
microalgae (Tetraselmis), yeast (Saccharomyces) and bacte-
taining diet (b-glu), b-glucan plus B. subtilis-containing
ria (Bacillus, Lactobacillus, Vibrio) have been used as bioac-
diet (b-glu+Bs), and b-glucan plus P. acidilactici-contain-
tive feed supplements or as a water additive (Austin et al.
ing diet (b-glu+Pa)] were fed to L. vannamei for 90 days.
1992; Gullian et al. 2004; Barnes et al. 2006; Ziaei-Nejada
Shrimp fed the b-glu and b-glu+Pa diets exhibited similar
et al. 2006; Balc azar et al. 2007; Wang 2007; Castex et al.
growth performance and body protein content, which
2009; Liu et al. 2009; Zhou et al. 2009; Leyva-Madrigal
were significantly higher than those of shrimp fed the con-
et al. 2011). Pediococcus acidilactici was shown to produce
trol diet (P < 0.05). No significant differences in haemol-
bacteriocins and has potential for use as a probiotic in fish;
ymph triglyceride, cholesterol, protein, haemolymph urea
however, information about its use as a probiotic in shrimp
nitrogen or chloride were detected among the experimen-
culture is scarce (Shelby et al. 2006; Anastasiadou et al.
tal diets. However, dietary b-glucan alone increased the
2008; Merrifield et al. 2011). Furthermore, a technique to
haemolymph glucose level and osmolarity (P < 0.05). Syn-
deliver viable probiotics to the shrimp gastrointestinal tract
biotic supplementation had greater effects on intestinal
needs to be developed (Solanki et al. 2013). Microencapsu-
microbiota and morphology than dietary b-glucan alone.
lation, which is a process used to pack probiotics within a
For example, b-glu+Bs increased the number of intestinal
shell material, may prove to be a practical method for
lactic acid bacteria and decreased the number of Vibrio
increasing the efficacy of probiotics.
spp. (P < 0.05), and b-glu+Pa increased the height of
Prebiotics are non-digestible food ingredients that are
intestinal villi.
selectively utilized by probiotics such as Lactobacillus and
Bifidobacterium. A particular arrangement of long chains
KEY WORDS: Bacillus subtilis, Litopenaeus vannamei, Pedio-
of monosaccharides in prebiotics causes them to be non-
coccus acidilactici, probiotics, shrimp, b-glucan
digestible by hydrolytic enzymes of the animal host (Ringo
et al. 2010). b-glucan, a homopolymer of glucose, has been
Received 22 May 2014; accepted 12 January 2015
isolated from a wide range of natural sources, including cell
Correspondence: S. Boonanuntanasarn, School of Animal Production
Technology, Institute of Agricultural Technology, Suranaree University
walls of bacteria, fungi, yeast and cereal grains, and used
of Technology, Nakhon Ratchasima 30000, Thailand. as a feed additive (Jaskari et al. 1998; Dalmo & Bøgwald
E-mail: surinton@sut.ac.th 2008). Dietary cosupplementation with probiotics and

..............................................................................................

ª 2015 John Wiley & Sons Ltd

prebiotics, which is defined as synbiotics, is a potentially use-
ful approach for providing feed additives to aquatic animals.
Despite the potential for synergistic effects, research into the
use of synbiotics as feed additives in shrimp is limited.
The Pacific white shrimp, Litopenaeus vannamei, is a
euryhaline species. Currently, it is the most common
shrimp species farmed throughout the world. In order to
meet rising global demand, shrimp cultivation has been
expanded not only to coastal regions but also to areas that
are supplied with inland brackish groundwater. The
demand for environmentally friendly sustainable aquacul-
ture will benefit from development of prebiotics, probiotics
and synbiotics as feed additives to improve shrimp produc-
tion. In this study, we investigated the effects of dietary
b-glucan and microencapsulated probiotics (Bacillus subtilis
or Pedicoccus acidilactici) on shrimp growth, whole body
composition, intestinal morphology and microbiota and
haemolymph constituents.
Bacillus subtilis and Pediococcus acidilactici were separately
cultured in a sterilized de Man, Rogosa and Sharpe (MRS)
agar supplemented with 3.0% NaCl (wt/vol) in a shaking
incubator at 30 °C for 16–18 h. After incubation, the cells
(a) (b)
(c) (d)
(e) (f) (g)
were harvested by centrifugation at 2000 g for 10 min and
washed twice with peptone water. Bacterial cells then were
observed with a scanning electron microscope (SEM)
(Fig. 1a,b). Microencapsulation of B. subtilis and P. acidi-
lactici was performed using the water-in-oil emulsion pro-
cess. Briefly, sticky rice-water that was enriched with
natural microbes was used to hydrolyse milk at 30 °C for
40 h. Then, the mixture of curd (280 g) and whey (720 g)
underwent NaOH hydrolysis at pH 9.0 for 3 h at 60 °C,
followed by adjusting pH to 7.0 with acetic acid and sterili-
zation by autoclaving. Each cell pellet (10 g) was mixed
with milk hydrolysate (75 g), and 0.9 mL of CaCl2 (10%
w/v) was added. The mixture was poured into sunflower oil
and stirred at 1400 rpm at 70 °C for 20 min. Microencap-
sulation of B. subtilis and P. acidilactici was analysed with
scanning electron microscope (SEM) (Fig. 1c,d).
The experimental design was completely randomized with
four treatment diets, each of which was replicated five
times. The four treatment diets were as follows: basal diet
(control, C), b-glucan-containing diet (b-glu), b-glucan plus
B. subtilis-containing diet (b-glu+BS) and b-glucan plus
P. acidilactici-containing diet (b-glu+Pa). Table 1 shows
the basal dietary ingredients and the proximate composi-
tion (moisture, crude protein, crude fat and ash content) of
Figure 1 Microencapsulation of Bacil-
lus subtilis and Pediococcus acidilactici.
Scanning electron microscopy (SEM)
images of B. subtilis (a), microencapsu-
lated B. subtilis (c), and inclusion of
microencapsulated B. subtilis in the diet
(e); SEM images of P. acidilactici (b),
microencapsulated P. acidilactici (d)
and inclusion of microencapsulated
P. acidilactici in the diet (f); SEM
image of the control diet (g). Scale bars
represent 5 lm (a, b) and 10 lm (c–g).
..............................................................................................
Aquaculture Nutrition ª 2015 John Wiley & Sons Ltd
 Table 1 Ingredients and proximal composition of the basal diet
Ingredient
Fish meal
Soybean meal
Wheat flour
Wheat gluten
Shrimp meal
Fish oil
Lecithin
Shrimp Premix1
1
Proximate composition (g kg dry weight)
Dry matter
Crude protein
Crude lipid
Crude fibre
Crude ash
1 mg/100 g premix: vitamin A (retinol) 7500 IU, vitamin D3 (cho-
lecalciferol) 1500 IU, vitamin E (tocopherol) 70.0, vitamin K3
(menadione) 12.0, vitamin B1 (thiamine) 30.0, vitamin B2 (ribofla-
vin) 25.0, vitamin B5 (pantothenic acid) 75.0, vitamin B6 (pyridox-
ine) 5000.0, nacin 50.0, vitamin C (ascorbic acid) 150.0, biotin 0.4,
folic acid 6.0, inositol 150.0, betain 0.005 %, copper sulfate 40.0,
zinc oxide 70.0, manganese oxide 18.0, selenium 0.25, iodine 1.0,
cobalt 0.08.
the experimental diets as determined following standard
AOAC methods (1990).
The b-glucan diet was prepared by adding BETA-S
(LEIBER) at 0.5 g kg 1 to the basal diet, which was
equivalent to b-glucan at 0.35 g kg 1. The dry ingredients
were ground using a hammer grinder. The mixture ingredi-
ents were extruded and steamed at 90 °C for 10 min. The
resulting pellets were dried at 90 °C for 2 h and stored at
4 °C until use. Experimental diets were generated by add-
ing the microencapsulated probiotics to the pellets at a
concentration of 5 9 107 CFU kg 1 of diet. The incorpo-
ration of microencapsulated probiotics in experimental
diets was confirmed using SEM (Fig. 1e,f).
Litopenaeus vannamei (postlarvae 5) were obtained from a
commercial farm in Chonburi, Thailand, and reared in the
hatchery at Suranaree University of Technology Farm (SUT
Farm) for 1 month. Shrimp were then graded by their size
uniformity (0.15  0.03 g mean initial weight) before sam-
pling. Five tanks (replicates) were randomly assigned to each
treatment diet, and 20 shrimp were randomly distributed
into each rectangular plastic tank with a volume of 100 L
under continuous aeration. Each tank was provided with an
internal filter system consisting of water flow through
sponge, oyster shell and bio-balls. To acclimatize the shrimp
..............................................................................................
Aquaculture Nutrition ª 2015 John Wiley & Sons Ltd
to the experimental conditions, the shrimp were fed the basal
g kg 1 diet for 2 weeks. Next, the experimental diets were provided
five times (9:00, 12:00, 15:00, 21:00 and 0:00) daily at the
220
ration of 5% body weight per day to all tanks for 90 days.
360
250 During the experiments, salinity was maintained at 10&.
60 Uneaten feed and faeces were removed, and sufficient
20 seawater was added to maintain 100 L in each tank. Air and
40
10 water temperatures were measured daily, and the dissolved
40 oxygen (DO) content and pH were determined weekly using
a DO meter and a pH meter, respectively. In addition, total
922
443
ammonia content was measured weekly using Tetratest
83 NH3/NH4+ (Tetra GmbH, Melle, Germany). Water temper-
36 ature (mean  SD) was 26.8  0.3 °C, and pH and DO con-
84
tent were within acceptable ranges (i.e. pH of 8.20  0.18;
DO of 6.47  0.64 mg L 1). Total ammonia content was
maintained at <0.25 mg L 1.
After the 90-day experimental period, shrimp in each tank
was counted and weighed to determine survival rate, final
body weight and feed efficiency (FE). Five shrimp from
each replicate tank were removed for haemolymph collec-
tion. A haemolymph sample (100 lL) was withdrawn from
the ventral sinus of each shrimp using a disposable syringe
containing 0.2 mL of anticoagulant solution (30 mM triso-
dium citrate, 340 mM sodium chloride, 10 mM EDTA,
120 mM glucose, pH 7.55, osmolality 480 mOsm kg 1). A
10 lL subsample of haemolymph was used to measure
osmotic pressure with a freezing-point osmometer (The
Fiske@210 Osmometer; Advanced Instruments, Inc., Nor-
wood, MA, USA). After haemolymph sampling, the intes-
tine of each shrimp was excised for histological study and
evaluation of the intestinal microbial population. In addi-
tion, after 45-day experimental period, two shrimp from
each tank were sampling for determination of intestinal his-
tology and microbial population. The specimens from each
replicate were pooled and frozen for proximate composi-
tion analysis of the whole body according to AOAC meth-
ods (1990).
To determine the effect of dietary b-glucan and B. subtilis
or P. acidilactici on intestinal morphology, two shrimp from
each replicate of each treatment were sampled and prepared
for histological study following Boonanuntanasarn et al.
(2014). Portions of the anterior and posterior intestine were
dissected and preserved in 10% phosphate-buffered
formalin at pH 7.2. After dehydration, the tissue was
embedded in a paraffin wax, cut into slices 5 lm thick and
mounted on glass slides. After deparaffinization, the slides
were dehydrated and stained with haematoxylin and eosin.
The height of the villi of stained sections was measured
under a microscope using an ocular micrometer at 1009
magnification.
mined using the cholesterol oxidase-phenol+aminophenaz-
one (CHOD-PAP) technique described by Flegg (1973).
Haemolymph protein concentrations were analysed using
the Biuret method (Gornall et al. 1949). BUN content was
measured using a modified indophenol colorimetric method
(Weatherburn 1967), and chloride concentration was esti-
mated using the thiocyanate method (Hamilton 1966).

After intestinal sampling for histological analysis, the
remaining intestinal samples with known weight were
aseptically pooled and homogenized in sterilized peptone
water (0.85% NaCl). Homogenized samples were diluted
serially with peptone water, and 100 lL aliquots was
spread-plated (in triplicate for each treatment) on a set of
five semiselective agars. Total counts of bacteria, Lactoba-
cilli, Bifidobacterium, fungi and Vibrio spp. were obtained
by plating on plate count agar (PCA), MRS agar, Raffi-
nose Bifidobacterium agar, sabouraud dextrose agar and
thiosulfate-citrate-bile salts-sucrose agar plates (TCBS),
respectively. Bacterial population numbers were recorded
as a logarithm of colony-forming units per gram of intes-
tine (Log CFU g 1).
Concentrations of the following haemolymph constituents
were measured: glucose, triglyceride, cholesterol, total pro-
tein, blood urea nitrogen (BUN) and chloride. Haemol-
ymph glucose content was measured by the glucose oxidase
method using a Ransod Kit (Randox, Crumlin, UK).
Triglyceride concentration was measured using the glyc-
erol-3-phosphate oxidase-sodium N-ethyl-N-(3-sulfopropyl)
m-anisidine (GPO-ESPAS) method described by Bucolo &
David (1973). Cholesterol content was quantitatively deter-
Statistical analysis was performed using SPSS for Win-
dows, version 10 (SPSS Inc., Chicago, IL, USA). All data
were analysed by one-way analysis of variance (ANOVA).
When significant differences were found among groups,
Duncan’s multiple range test was conducted to rank.
Throughout the experiment, effects and differences were
declared to be significant at P < 0.05.
Table 2 shows the growth performance and survival rates
of experimental shrimp. Administration of the b-glu and b-
glu+Pa diets for 90 days resulted in significantly higher
(P < 0.05) final weight, weight gain and body length com-
pared to values for the control group. Supplementation
with b-glucan and B. subtilis did not significantly result in
a higher body weight compared to the control group. There
were no significant differences in feed efficiency (FE) and
survival rate among the experimental treatments.
Table 3 showed the analysis of proximate chemical com-
position of the experimental shrimp meat. Comparing to
the control shrimp, shrimp fed b-glu or b-glu and probiot-
ics had higher moisture content (P < 0.05). In addition, sig-
nificant increase in protein content in meat was observed in
shrimp fed b-glu and b-glu+Pa diets (P < 0.05). Only
shrimp fed the b-glu+Bs diet had higher ash content in the

Table 2 Growth performance of Litopenaeus vannamei fed with experimental diets1

Experimental diets

Parameters C b-glu b-glu+Bs b-glu+Pa

Initial weight (g) 0.15  0.02 0.15  0.02 0.16  0.02 0.16  0.02
Final weight (g) 3.45  0.36a 5.30  0.73b 4.51  0.41ab 5.20  0.38b
Weight gain2 (g) 3.29  0.36a 5.16  0.74b 4.36  0.42ab 5.04  0.38b
Total length (cm) 7.66  0.26a 9.14  0.25b 8.35  0.48ab 9.28  0.08b
FE3 0.37  0.04 0.54  0.08 0.45  0.05 0.51  0.04
Survival (%) 80.0  5.7 80.0  3.5 77.1  7.3 85.7  7.8
1
Values are means  SD of five replicates. Means with a different superscript in each row differed significantly from each other
(P < 0.05).
2
Weight gain = final mean body weight initial mean body weight.
3
Feed efficiency (FE) = wet weight gain 9 dry feed fed 1.
..............................................................................................

Aquaculture Nutrition ª 2015 John Wiley & Sons Ltd

 Table 3 Proximate compositions of Litopenaeus vannamei (g kg 1) chloride were detected among the experimental groups.
that were fed the experimental diets (mean  SD, n = 5)1 Shrimp fed the b-glu diet had the highest osmolarity com-
Moisture Protein Fat Ash pared to the control group (P < 0.05).
(g kg 1) (g kg 1) (g kg 1) (g kg 1) Intestinal morphology was also investigated at 45 and
C 798.9  4.3a 111.3  1.5b 4.9  1.3 27.6  2.1b 90 days (Table 5). At 45 days, shrimp fed the b-glu+Pa diet
b-glu 765.7  3.7c 130.0  7.8a 5.4  1.5 31.4  5.6ab had the highest intestinal villi height in both the proximal
b-glu+Bs 776.1  4.1b 122.4  10.3ab 6.5  1.2 35.6  0.9a and distal parts of the intestine (P < 0.05). At 90 days,
b-glu+Pa 762.6  7.9c 130.5  8.5a 6.3  0.9 29.4  3.7b
shrimp fed the b-glu+Pa and b-glu+Bs diets had higher
1
Means with different superscripts in each column differed sig- intestinal villi height in the proximal part than that of
nificantly from each other (P < 0.05).
shrimp fed Diet C. Furthermore, only shrimp fed the b-
glu+Pa diet had higher intestinal villi height in the distal
meat compared with shrimp fed the control diet (P < 0.05). part compared with shrimp fed the control diet (P < 0.05).
No significant difference in fat content among the groups
was observed.
The population of microbiota in the intestines of experi-
mental shrimp was evaluated at 45 and 90 days (Table 4). Although the use of probiotics in shrimp aquaculture has
Throughout the experimental period, total bacterial count been studied previously (Chiu et al. 2007; Wang et al.
was similar among the experimental groups. For both time 2012; Sapcharoen & Rengpipat 2013), little work has
points, shrimp fed the b-glu+Bs diet had the highest num- focused on designing an approach to protect probiotics
ber of lactic acid bacteria. The number of intestinal fungi during the ingestion and passage into the shrimp gastroin-
and Bifidobacterium was similar among the experimental testinal tract (Wongsasak et al. 2015). This study demon-
groups. At 45 days, shrimp fed the b-glu+Bs and b-glu+Pa strated that microencapsulation of probiotics (Fig. 1) can
diets contained significantly fewer Vibrio bacteria compared enhance the delivery of probiotics into the digestive tract of
with shrimp fed the control diet (P < 0.05). At 90 days, shrimp. The effects of supplementation with the prebiotic
only shrimp fed the b-glu+Bs diet contained significantly b-glucan alone and with synbiotics consisting of b-glucan
fewer intestinal Vibrio bacteria compared to shrimp fed the with microencapsulated B. subtilis or P. acidilactici were
control diet (P < 0.05). investigated to provide useful data for both biological
Figure 2 shows the haemolymph chemical parameters of research and improved shrimp production.
experimental shrimp. The glucose in haemolymph fed Dietary b-glucan has been shown to have variable effects
shrimp fed b-glu or b-glu and probiotics did not signifi- on growth performance in shrimp. For example, the Pacific
cantly differ from that of the shrimp fed control diet. How- white shrimp that were fed dietary yeast glucan for 4 weeks
ever, supplementation of b-glu and probiotics resulted in a showed no significant growth enhancement (Burgents et al.
lower glucose compared to the supplementation of b-glu 2004; Chotikachinda et al. 2008). In contrast, tiger shrimp
alone (P < 0.05). No significant differences in cholesterol, treated with b-glucan by immersion for 43 days exhibited
triglyceride, total protein, haemolymph urea nitrogen and enhanced growth (Sung et al. 1994). The present results

Table 4 Microbiota population in the intestine of Litopenaeus vannamei that were fed the experimental diets (mean  SD, n = 5)1

Total count LAB Fungi Bifidobacterium Vibrio

log (CFU g 1) log (CFU g 1) log (CFU g 1) log (CFU g 1) log (CFU g 1)

45 days
C 6.6  0.9 5.2  0.7b 5.5  0.7 5.7  0.7 6.3  0.7a
b-glu 5.9  1.2 5.5  0.5ab 5.5  0.9 6.2  0.9 6.1  0.5a
b-glu+Bs 6.5  0.6 6.5  0.8a 6.2  1.3 6.5  0.6 5.2  0.4b
b-glu+Pa 6.3  0.4 5.9  1.1ab 5.4  1.2 6.4  1.0 5.1  0.8b
90 days
C 6.2  0.7 4.8  0.7b 5.6  0.4 5.5  0.9 6.1  0.6a
b-glu 6.0  0.5 4.9  0.4b 4.9  0.9 5.4  0.7 5.4  0.5a
b-glu+Bs 6.7  0.8 6.1  1.0a 5.6  1.0 6.3  0.7 4.5  0.4b
b-glu+Pa 5.9  0.4 5.4  0.5ab 5.0  0.6 5.5  0.6 5.5  0.7a
1
Means with different superscripts in each column differed significantly from each other (P < 0.05).
..............................................................................................

Aquaculture Nutrition ª 2015 John Wiley & Sons Ltd

 0.5 0.3

Cholesterol (mmol L–1)

Glucose (mmol L–1) a
ab
0.4 b b 0.2

0.3 0.1

0.2 0
Control β-Glu β-Glu+ β-Glu+ Control β-Glu β-Glu+ β-Glu+
Bs Pa Bs Pa

60 100
Triglyceride (μmol L–1)

Total protein (mg L–1)

75
40
50
20
25

0 0
Control β-Glu β-Glu+ β-Glu+ Control β-Glu β-Glu+ β-Glu+
Bs Pa Bs Pa

1.00 40
Chloride (mmol L–1)
BUN (mmol L–1)

0.75 30

0.50 20

0.25 10

0.00 0
Control β-Glu β-Glu+ β-Glu+ Control β-Glu β-Glu+ β-Glu+
Bs Pa Bs Pa

1500
Osmolarity (mosm kg–1)

a
ab ab
b
1250 Figure 2 Haemolymph constituents of
Litopenaeus vannamei that were fed the
experimental diets for 90 days. The dif-
1000
ferent letters denote significant differ-
Control β-Glu β-Glu+ β-Glu+ ences in haemolymph constituents
Bs Pa among experimental diets at P < 0.05.

showed that higher growth was observed in shrimp fed the in numerous studies. For instance, Artemia enriched with
b-glucan alone. These data indicate that the effects of P. acidilactici improved weight gain of Pollock larvae
b-glucan on growth might be species specific and/or depend (Gatesoupe 2002). In contrast, the use of P. acidilactici as
on the b-glucan source, administration method and dura- probiotic supplementation did not improve growth perfor-
tion of supplementation. In addition, neither dietary mance in rainbow trout (Aubin et al. 2005; Merrifield et al.
b-glu+Pa nor b-glu+Bs significantly improved shrimp 2011), Nile tilapia (Shelby et al. 2006) or channel catfish
growth compared to dietary b-glucan alone, suggesting that (Shelby et al. 2007).
no synbiotic effect on growth enhancement in shrimp. Bacillus subtilis has been used widely as a probiotic in
Our results showed that cosupplementation with the pre- shrimp culture (Balc azar et al. 2007; Liu et al. 2009; Sap-
biotic b-glucan and the microencapsulated probiotic charoen & Rengpipat 2013). In our study, cosupplementa-
P. acidilactici had significant beneficial effects on growth of tion with b-glucan and B. subtilis resulted in a non-
L. vannamei. Although reports of the application of significant increase in growth compared to the control diet.
P. acidilactici as a probiotic in shrimp aquaculture are lim- There have been reports of different effects on growth
ited, its use as a probiotic in fish culture has been described response among different B. subtilis strains. For example,
..............................................................................................

Aquaculture Nutrition ª 2015 John Wiley & Sons Ltd

 Table 5 Intestinal villi height in portions of the intestines of supplementation with the b-glu+Pa or b-glu diets increased
Litopenaeus vannamei that were fed the experimental diets the protein composition. In addition, cosupplementation
(mean  SD, n = 5)1
with b-glucan and B. subtilis increased the ash content.
45 days 90 days Although the differences in proximate composition might
Proximal Distal Proximal Distal
be partly due to uncertainty values of the analytical
Diet part (µm) part (µm) part (µm) part (µm) method, the present results probably demonstrated the
positive effects of dietary b-glucan alone or b-glucan and
C 31.0  1.5b 31.5  3.1b 39.6  0.9b 39.5  3.1b
b-glu 32.2  5.5ab 37.1  5.0ab 37.4  5.5b 42.0  3.4ab probiotics on protein and ash contents in shrimp meat.
b-glu+Bs 37.7  4.7a 39.7  5.8ab 44.3  1.3a 39.4  3.1b Dietary prebiotics can modulate the microbial popula-
b-glu+Pa 38.6  3.7a 44.3  7.0a 47.7  1.9a 45.0  3.7a tion in the intestine of the host. For example, Jaskari et al.
1
Means with different superscripts in each column differed sig- (1998) reported that b-glucan provided substrates for the
nificantly from each other (P < 0.05). growth of Lactobacillus and Bifidobacterium in vitro. How-
ever, the prebiotic effects in vivo might be different. The
present study showed that supplementation with b-glucan
supplementation with B. subtilis UTM 126, E20 and BS11 did not significantly affect lactic acid bacteria, fungi and
had beneficial effects on growth responses in L. vannamei Bifidobacterium. Similarly, dietary fructooligosaccharide did
(Balcazar et al. 2007; Liu et al. 2009; Sapcharoen & not increase the numbers of intestinal Lactobacillus in L
Rengpipat 2013), whereas supplementation with B. subtilis vannamei (Zhou et al. 2007). Burgents et al. (2004) reported
BP11 did not result in significant growth enhancement that a yeast culture food supplement increased the survival
compared to shrimp fed the diet without probiotics (Sap- of L. vannamei that were challenged with Vibrio spp. In
charoen & Rengpipat 2013). Overall, these results demon- our study, dietary b-glucan decreased the intestinal Vibrio
strated variable effects of different probiotic strains on population, although the change was not statistically signif-
growth performances in aquatic animals. icant. Cosupplementation with b-glucan and B. subtilis led
Haemolymph metabolites have been shown to reflect to markedly increased numbers of lactic acid bacteria and
nutritional in shrimp (Pascual et al. 2003, 2004). Except for decreased numbers of Vibrio spp., demonstrating that die-
haemolymph glucose and osmolarity, we found that the b- tary the prebiotic and probiotics were able to exclude path-
glu, b-glu+Pa, and b-glu+Bs diets had no effects on the levels ogenic bacteria. These observations were in agreement with
of most examined metabolites, including cholesterol, triglyc- reports for other probiotics. For example, dietary supple-
eride, protein, haemolymph urea nitrogen and chloride. Our mentations with P. acidilactici significantly increased the
results showed that shrimp fed dietary b-glucan alone had lactic acid bacteria in red tilapia (Ferguson et al. 2010).
the highest haemolymph glucose level, which indicated that Moreover, supplementation with probiotics increased the
they digested the b-glucan. Thanardkit et al. (2002) reported survival of L. vannamei infected with Vibrio spp. (Balc azar
that dietary b-glucan induced the activity of b-glucanase in et al. 2007; Luis-Villase~nor et al. 2013). In our study, no
P. monodon. Compared with dietary b-glucan alone, shrimp significant differences in total bacterial count and numbers
fed the b-glu+Pa and b-glu+Bs diets had lower blood glu- of fungi and Bifidobacterium were detected among the
cose, suggesting that the digested glucose was utilized by experimental groups. The differences in the microflora pop-
both probiotics. Shrimp fed the b-glu diet had the highest ulation induced by dietary supplements may occur at the
osmolarity, and shrimp fed the b-glu+Bs and b-glu+Pa diets species level, which will require special techniques to study.
tended to have higher osmotic concentration than shrimp fed Increased villi length may increase the ability to absorb
the control diet. Osmolarity is defined as the osmoles of sol- digested nutrients, thereby providing the potential to
ute particles in haemolymph. These results suggest that die- enhance growth performance in animals. At both 45
tary supplementation with b-glucan and probiotics could and 90 days, cosupplementation with b-glucan and
increase osmotic concentration, which would benefit shrimp P. acidilactici increased the villi height in both the proxi-
culture in low-salinity water. mal and distal areas of the intestine. The highest intesti-
Supplementation with probiotics has been shown to nal villi height was probably related to the highest body
increase digestive enzyme activities (Wang 2007; Zhou weight in shrimp fed on Diet b-glu+Pa. Our result was
et al. 2009), which would contribute to enhance nutrient in an agreement with the observation in rainbow trout
assimilation and accumulation in body. While the b-glu, b- fed with dietary P. acidilactici. Dietary P. acidilactici
glu+Pa and b-glu+Bs diets did not alter body lipid content, improved microvilli in the proximal part of intestine in
..............................................................................................

Aquaculture Nutrition ª 2015 John Wiley & Sons Ltd

rainbow trout although this effect was not detected in
distal part of intestine (Merrifield et al. 2010). Abid et al.
(2013) reported that supplementation with a synbiotic
that included P. acidilactici and FOS improved the mi-
crovilli in rainbow trout. In contrast, supplementation
with P. acidilactici did not increase villi height in red tila-
pia (Ferguson et al. 2010). Our results showed that sup-
plementation with b-glucan alone did not significantly
increase the villi height compared to the control group.
This observation was not in accordance with previous
results in chickens, for which b-glucan had positive effects
on intestinal villi (Morales-Lopez et al. 2009; Shao et al.
2013). We also found that supplementation with b-glucan
and B. subtilis increased the villi height, although this
effect was observed only in the proximal part of intestine.
In rainbow trout, B. subtilis did not significantly improve
intestinal villi height (Merrifield et al. 2010). These data
indicate that the effects of supplementation with b-glucan
and probiotics on intestinal morphology would be related
to different intestinal microbiota which vary among
different animals.
In conclusion, supplementation with b-glucan alone or b-
glucan together with P. acidilactici had positive effects on
shrimp growth and protein composition in the meat. More-
over, dietary b-glucan increased the haemolymph glucose
and osmotic concentrations. Synbiotic supplementation had
greater effects on intestinal microbiota and morphometry
than dietary b-glucan alone.
This study was supported by grants from Suranaree Uni-
versity of Technology, the Higher Education Research Pro-
motion and National Research University Project of
Thailand, Office of the Higher Education Commission, and
the National Research Council of Thailand (SUT3-303-50-
12-16). This work was also supported by the Thailand
Research Fund-Master Research Grants-Research and
Researchers for Industries (RRI) and Nuevotec Company
Limited [MRG545S040]. We acknowledge the help of Mr.
Sunai Plymee (SUT Farm) for maintaining the shrimp
throughout the study.
Abid, A., Davies, S.J., Waines, P., Emery, M., Castex, M.,
Gioacchini, G., Carnevali, O., Bickerdike, R., Romero, J. &
Merrifield, D.L. (2013) Dietary synbiotic application modulates
Atlantic salmon (Salmo salar) intestinal microbial communities
and intestinal immunity. Fish Shellfish Immunol., 12, 97–109.
Anastasiadou, S., Papagianni, M., Filiousis, G., Ambrosiadis, I. &
Koidis, P. (2008) Pediocin SA-1, an antimicrobial peptide from
Pediococcus acidilactici NRRL B5627: production conditions,
purification and characterization. Bioresour Technol., 99, 5384–
5390.
AOAC (1990) Association of Official Analytical Chemists. Official
Methods of Analysis of the Association of Official Analytical
Chemists, Vol. 1, 14th edn. AOAC, Arlington, VA, USA.
Aubin, J., Gatesoupe, E.-J., Labbe, L. & Lebrun, L. (2005) Trial
of probiotics to prevent the vertebral column compression syn-
drome in rainbow trout (Onchorhynchus mykiss Walbaun). Aqua-
cult. Res., 36, 758–767.
Austin, B., Baudet, E. & Stobie, M. (1992) Inhibition of bacterial
fish pathogens by Tetraselmis suecica. J. Fish Dis., 15, 55–61.
Balcazar, J.L., Rojas-Luna, T. & Cunningham, D.P. (2007) Effect
of the addition of four potential probiotic strains on the survival
of pacific white shrimp (Litopenaeus vannamei) following immer-
sion challenge with Vibrio parahaemolyticus. J. Invertebr. Pathol.,
96, 147–150.
Barnes, M.E., Durben, D.J., Reeves, S.G. & Sanders, R. (2006)
Dietary yeast culture supplementation improves initial rearing of
McConaughy strain rainbow trout. Aquacult. Nutr., 12, 388–394.
Boonanuntanasarn, S., Khaomek, P., Pitaksong, T. & Hua, Y.
(2014) The effects of supplementation of activated charcoal on
the growth, health status and fillet compostion-odor of Nile tila-
pia (Oreochromis niloticus) before harvesting. Aquacult. Int., 22,
1417–1436.
Bucolo, G. & David, H. (1973) Quantitative determination of serum
triglycerides by the use of enzymes. Clin. Chem., 19, 476–482.
Burgents, J.E., Burnett, K.G. & Burnett, L.E. (2004) Disease resis-
tance of Pacific white shrimp, Litopenaeus vannamei, following
the dietary administration of a yeast culture food supplement.
Aquaculture, 231, 1–8.
Castex, M., Lemaire, P., Wabete, N. & Chim, L. (2009) Effect of
dietary probiotic Pediococcus acidilactici on antioxidant defenses
and oxidative stress status of shrimp Litopenaeus stylirostris.
Aquaculture, 294, 306–313.
Chiu, C.H., Guu, Y.K., Liu, C.H., Pan, T.M. & Cheng, W. (2007)
Immune responses and gene expression in white shrimp, Litope-
naeus vannamei, induced by Lactobacillus plantarum. Fish Shell-
fish Immunol., 23, 364–377.
Chotikachinda, R., Lapjatupon, W., Chaisilapasung, S., Sangsue,
D. & Tantikitti, C. (2008) Effect of inactive yeast cell wall on
growth performance, survival rate and immune parameters in
Pacific White Shrimp (Litopenaeus vannamei). Songklanakarin J.
Sci. Technol., 30, 687–692.
Dalmo, R.A. & Bøgwald, J. (2008) b-glucans as conductors of
immune symphonies. Fish Shellfish Immunol., 25, 384–396.
FAO/WHO (2001) Report of a Joint FAO/WHO expert consulta-
tion on evaluation of health and nutritional properties of probi-
otics in food including powder milk with live lactic acid bacteria.
In: Health and Nutritional Properties of Probiotics in Food
Including Powder Milk with Live Lactic Acid Bacteria. FAO/
WHO, Cordoba, Argentina.
Ferguson, R.M.W., Merrifield, D.L., Harper, G.M., Rawling,
M.D., Mustafa, S., Picchietti, S., Balcazar, J.L. & Davies, S.J.
(2010) The effect of Pediococcus acidilactici on the gut microbi-
ota and immune status of on-growing red tilapia (Oreochromis
niloticus). J. Appl. Microbiol., 109, 851–862.
Flegg, H.M. (1973) An investigation of determination of serum cho-
lesterol by an enzymatic method. Ann. Clin. Biochem., 10, 79–84.
Fuller, R. (1989) Probiotics in man and animals. J. Appl. Bacte-
riol., 66, 365–378.
..............................................................................................
Aquaculture Nutrition ª 2015 John Wiley & Sons Ltd
 Gatesoupe, F.J. (2002) Probiotic and formaldehyde treatments of
Artemia nauplii as food for larval pollack, Pollachius pollachius.
Aquaculture, 212, 347–360.
Gornall, A.G., Bardawill, C.J. & David, M.M. (1949) Determina-
tion of serum proteins by means of the biuret reaction. J. Biol.
Chem., 177, 751–766.
Gullian, M., Thompson, F. & Rodriguez, J. (2004) Selection of
probiotic bacteria and study of their immunostimulatory effect
in Penaeus vannamei. Aquaculture, 233, 1–14.
Hamilton, R.H. (1966) A direct photometric method for chloride
in biological fluids, employing mercuric thiocyanate and per-
chloric acid. Clin. Chem., 11, 1–17.
Jaskari, J., Kontula, P., Siitonen, A., Jousimies-Somer, H., Matti-
la-Sandholm, T. & Poutanen, K. (1998) Oat b-glucan and xylan
hydrolysates as selective substrates for Bifidobacterium and Lac-
tobacillus strains. Appl. Microbiol. Biotechnol., 49, 175–181.
Leyva-Madrigal, K.Y., Luna-Gonzalez, A., Escobedo-Bonilla,
C.M., Fierro-Coronado, J.A. & Maldonado-Mendoza, I.E.
(2011) Screening for potential probiotic bacteria to reduce preva-
lence of WSSV and IHHNV in whiteleg shrimp (Litopenaeus
vannamei) under experimental conditions. Aquaculture, 322, 16–22.
Liu, C.H., Chiu, C.S., Ho, P.L. & Wang, S.W. (2009) Improve-
ment in the growth performance of white shrimp, Litopenaeus
vannamei, by a protease-producing probiotic, Bacillus subtilis
E20, from natto. J. Appl. Microbiol., 107, 1031–1041.
Luis-Villase~nor, I.E., Castellanos-Cervantes, T., Gomez-Gil, B.,
Carrillo-Garcıa, A.E., Campa-C ordova, A.I. & Ascencio, F.
(2013) Probiotics in the intestinal tract of juvenile whiteleg
shrimp Litopenaeus vannamei: modulation of the bacterial com-
munity. World J. Microbiol. Biotechnol., 29, 257–265.
Merrifield, D.L., Harper, G., Dimitroglou, A., Ringo, E. & Davies,
S.J. (2010) Possible influence of probiotic adhesion to intestinal
mucosa on the activity and morphology of rainbow trout (On-
corhynchus mykiss) enterocytes. Aquacult. Res., 41, 1268–1272.
Merrifield, D.L., Bradley, G., Harper, G.M., Baker, R.T.M.,
Munn, C.B. & Davies, S.J. (2011) Assessment of the effects of
vegetative and lyophilized Pediococcus acidilactici on growth,
feed utilization, intestinal colonization and health parameters of
rainbow trout (Oncorhynchus mykiss Walbaum). Aquacult. Nutr.,
17, 73–79.
Morales-L opez, R., Auclair, E., Garcıa, F., Esteve-Garcia, E. &
Brufau, J. (2009) Use of yeast cell walls; b-1,3/1, 6-glucans; and
mannoproteins in broiler chicken diets. Poult. Sci., 88, 601–607.
Pascual, C., Gaxiola, G. & Rosas, C. (2003) Blood metabolites
and hemocyanin of the white shrimp, Litopenaeus vannamei: the
effect of culture conditions and a comparison with other crusta-
cean species. Mar. Biol., 142, 735–745.
Pascual, C., Zenteno, E., Cuzon, G., Sanchez, A., Gaxiola, G.,
Taboada, G., Suarez, J., Maldonado, T. & Rosas, C. (2004)
Litopenaeus vannamei juveniles enegetic balance and immunolog-
ical response to dietary protein. Aquaculture, 236, 431–450.
Ringo, E., Olsen, R.E., Gifstad, T.Ø., Dalmo, R.A., Amlund, H.,
Hemre, G.-I. & Bakke, A.M. (2010) Prebiotics in aquaculture: a
review. Aquacult. Nutr., 16, 117–136.
..............................................................................................
Aquaculture Nutrition ª 2015 John Wiley & Sons Ltd
Sapcharoen, P. & Rengpipat, S. (2013) Effects of the probiotic
Bacillus subtilis (BP11 and BS11) on the growth and survival of
Pacific white shrimp, Litopenaeus vannamei. Aquacult. Nutr., 19,
946–954.
Shao, Y., Guo, Y. & Wang, Z. (2013) b-1,3/1,6-Glucan alleviated
intestinal mucosal barrier impairment of broiler chickens
chal1lenged with Salmonella enterica serovar Typhimurium.
Poult. Sci., 92, 1764–1773.
Shelby, R., Lim, C., Yildirm-Aksoy, M. & Delaney, M. (2006)
Effects of probiotic diet supplements in disease resistance and
immune response of young Nile Tilapia (Oreochromis niloticus).
J. Appl. Aquacult., 18, 22–34.
Shelby, R., Lim, C., Yildirm-Aksoy, M. & Klesius, P.H. (2007)
Effects of probiotic bacteria as dietary supplements on growth
and disease resistance in young channel catfish, Ictalurus puncta-
tus (Rafinesque). J. Appl. Aquacult., 19, 81–91.
Solanki, H.K., Pawar, D.D., Shah, D.A., Prajapati, V.D., Jani,
G.K., Mulla, A.M. & Thakar, P.M. (2013) Development of
microencapsulation delivery system for long-term preservation of
probiotics as biotherapeutics agent. Biomed. Res. Int., 2013,
1–21.
Sung, H.H., Kuo, G.H. & Song, Y.L. (1994) Vibriosis resistance
induced by glucan treatment in tiger shrimp (Penaeus monodon).
Fish Pathol., 29, 11–17.
Thanardkit, P., Khunrae, P., Suphantharika, M. & Verduyn, C.
(2002) Glucan from spent brewer’s yeast: preparation, analysis
and use as a potential immunostimulat in shrimp feed. World J.
Microbiol. Biotechnol., 18, 527–539.
Wang, Y.B. (2007) Effect of probiotics on growth performance
and digestive enzyme activity of the shrimp Penaeus vannamei.
Aquaculture, 269, 259–264.
Wang, Y., Fu, L. & Lin, J. (2012) Probiotic (Bacillus coagulans)
cells in the diet benefit the white shrimp Litopenaeus vannamei.
J. Sea Res., 31, 855–860.
Weatherburn, M.W. (1967) Phenol-hypochlorite reaction for deter-
mination of ammonia. Anal. Chem., 39, 971.
Wongsasak, U., Chaijumrak, S., Kamkong, S. & Boonanuntana-
sarn, S. (2015) Effects of dietary supplementation with b-glucan
and synbiotics on immune gene expression and immune parame-
ters under ammonia stress in the Pacific white shrimp. Aquacul-
ture, 436, 179–187.
Zhou, Z., Ding, Z. & Huiyuan, L.V. (2007) Effects of dietary
short-chain fructooligosaccharides on intestinal microflora, sur-
vival, and growth performance of juvenile white shrimp, Litope-
naeus vannamei. J. World Aquacult. Soc., 38, 296–301.
Zhou, X., Wang, Y.B. & Lia, W.F. (2009) Effect of probiotic on
larvae shrimp (Penaeus vannamei) based on water quality, sur-
vival rate and digestive enzyme activities. Aquaculture, 287, 349–
353.
Ziaei-Nejada, S., Rezaeib, M.H., Takamic, G.A., Lovettd, D.L.,
Mirvaghefia, A.R. & Shakourie, M. (2006) The effect of Bacillus
spp. bacteria used as probiotics on digestive enzyme activity, sur-
vival and growth in the Indian white shrimp Fenneropenaeus in-
dicus. Aquaculture, 252, 516–524.